Multienzyme Cascade Bioreactor for a 10 min Digestion of Genomic DNA into Single Nucleosides and Quantitative Detection of Structural DNA Modifications in Cellular Genomic DNA

Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8–24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.